art39844_am.pdf - Deep Blue Repositories

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leDeletion of Macrophage Migration Inhibitory Factor Reduces Severity of Osteoarthritis in

Aged Mice

Meredith A. Rowe1, Lindsey R. Harper

2, Margaret A. McNulty

3, Anthony G. Lau

4, Cathy S.

Carlson2, Lin Leng

5, Richard J. Bucala

5, Richard A. Miller

6, Richard F. Loeser

1

Running title: Mif Deletion Reduces Age-related OA

This work was supported by grants from the National Institute on Aging (AG046990) to M.A.R.,

(AG044185) to R.F.L., and (AG024824) to R.A.M. and from the National Institute of Arthritis

and Musculoskeletal and Skin Diseases (AR049610) to R.J.B.

1Meredith A. Rowe, PhD and Richard F. Loeser, MD: Wake Forest School of Medicine,

Winston-Salem, North Carolina and University of North Carolina at Chapel Hill, Chapel Hill,

North Carolina. 2Lindsey R. Harper, BS and Cathy S. Carlson, DVM, PhD: University of

Minnesota, St. Paul, Minnesota. 3Margaret A. McNulty, PhD: Louisiana State University, Baton

Rouge, Louisiana. 4Anthony G. Lau, PhD: University of North Carolina at Chapel Hill, Chapel

Hill, North Carolina. 5Lin Leng, PhD and Richard J. Bucala, MD, PhD: Yale University, New

Haven, Connecticut. 6Richard A. Miller, MD, PhD: University of Michigan, Ann Arbor,

Michigan.

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This is the author manuscript accepted for publication and has undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process, which may lead to differencesbetween this version and the Version record. Please cite this article as doi:10.1002/art.39844.

This article is protected by copyright. All rights reserved.

http://dx.doi.org/10.1002/art.39844

http://dx.doi.org/10.1002/art.39844

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Address correspondence to: Richard F. Loeser M.D., Thurston Arthritis Research Center,

University of North Carolina, 3300 Thurston Building, CB #7280, Chapel Hill, NC 27599-7280.

E-mail: [email protected]

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Abstract

Objective: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine

that is elevated in the serum and synovial fluid of osteoarthritic (OA) patients. In this study, the

potential role of MIF in OA was studied using human joint tissues and in vivo in mice with age-

related and surgically induced OA.

Methods: MIF in conditioned media from human chondrocytes and meniscal cells and

from cartilage explants was measured by ELISA. Severity of OA was analyzed histologically in

male wild-type and Mif-/- mice at 12- and 22-months of age and following destabilization of the

medial meniscus (DMM) surgery in 12-week old Mif-/- mice as well as in wild-type mice treated

with a neutralizing MIF antibody. Synovial hyperplasia was graded in S100A8 immunostained

histologic sections. Bone morphometric parameters were measured by microCT analysis.

Results: Human OA chondrocytes secreted 3-fold higher levels of MIF than normal

chondrocytes, while normal and OA meniscal cells produced equivalent amounts. Compared to

age- and strain-matched controls, the cartilage, bone, and synovium in older adult mice with Mif

deletion were protected from changes of naturally occurring age-related OA. No protection from

DMM-induced OA was seen in young adult Mif-/- mice or in wild-type mice treated with anti-

MIF. Increased bone density in 8 week-old mice with Mif deletion was not maintained at 12-

months.

Conclusions: These results demonstrate a differential mechanism in the pathogenesis of

naturally occurring age-related OA compared to injury-induced OA. The inhibition of MIF may

represent a novel therapeutic target in the reduction of age-related OA.

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Aging has been well-described as a key risk factor for the development of osteoarthritis

(OA) (1). While aging does not directly cause OA, aging changes in joint tissues and possibly

circulating factors that change with age increase the susceptibility for the development of the

disease. Aging changes are reflected in the cartilage matrix including thinning of the articular

cartilage (2) and the accumulation of advanced glycation end products (AGE) which alters the

biomechanical properties of the joint (3). Aging changes are also evident in chondrocytes, where

chondrocytes isolated from aged individuals are more resistant to stimulation with the growth

factors insulin-like growth factor 1 (IGF-1) (4, 5), transforming growth factor-β (TGF-β) (6), and

bone morphogenetic protein-6 (BMP-6) (7) than chondrocytes isolated from younger individuals.

Inflammatory mediators have been found to be up-regulated in aged joint tissues including IL-7

in chondrocytes and synovial fluid in humans and IL-33, CXCL13, CCL8, and CCL5 in the

mouse knee joint (reviewed in Greene and Loeser (8)). The increased expression of these

inflammatory mediators, which is compounded by aging changes in the tissues, may contribute

to the development of OA.

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that has been

studied for its role in the immune system. MIF has been shown to function as a cytokine that

signals through the CD74 receptor (9) to increase neutrophil migration to regions of

inflammation and to promote the innate immune response (10, 11). Additionally, MIF promotes

macrophage activation, increasing phagocytosis and destruction of pathogens (11, 12). MIF has

been studied in autoimmune diseases including rheumatoid arthritis (RA) and systemic lupus

erythematosus (SLE). It has been shown to promote MMP-1 and MMP-3 production in synovial

fibroblasts isolated from the knee joints of RA patients (13) and also has been shown to be

significantly elevated in RA synovial fluid (14). In a more recent study, inhibition of MIF

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function either by deletion of Mif, or its receptor, Cd74, was found to reduce the severity of

disease in an RA model by reducing both inflammation and bone erosion (15). In SLE, the

amount of MIF in serum is positively correlated with tissue damage (16), and the renal

expression of MIF was shown to be significantly elevated in an SLE-prone mouse model (17).

The level of MIF was noted to be elevated in the serum and synovial fluid of patients with knee

OA compared to healthy controls (14, 18). However, the potential contribution of MIF to the

development of OA has not been previously studied.

Recently, Mif-/- 129Sv/C57Bl/6 mice were reported to have an increased lifespan when

compared to wild-type (WT) controls, suggesting a role for MIF in the aging process (19),

although more recent data (Miller, unpublished) suggest that this beneficial effect may be absent

on other genetic backgrounds. Like OA, atherosclerosis is a common disease of aging and

inhibition of MIF has been found to reduce atherosclerosis in a mouse model of the disease (20)

suggesting that MIF may represent a therapeutic target for age-related disease. Given the

association between aging and OA, we sought to determine the contribution of MIF to the

development of OA. We found that genetic disruption of Mif decreased the severity of naturally-

occurring OA in aged mice but not in a surgically-induced model of OA in younger adult mice.

MATERIALS & METHODS

Chondrocyte and meniscal cell isolation and culture. Normal human donor tissue

(both knee and ankle joints) was obtained from the Gift of Hope Organ and Tissue Donor

Network (Elmhurst IL) through the Rush University Medical Center in Chicago, IL.

Osteoarthritic tissue was obtained as surgical waste from total knee replacement surgeries

performed in the Department of Orthopaedic Surgery at Wake Forest Baptist Health and at the

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University of North Carolina Hospitals. The use of human tissue was approved by the

Institutional Review Boards at Rush University, Wake Forest Baptist Health, and the University

of North Carolina at Chapel Hill. All human donor tissue was de-identified before receipt, and

the cartilage and meniscus were processed separately. The cartilage was dissected away from

the subchondral bone in small flakes and rinsed in serum-free DMEM-F12 media (Gibco Life

Technologies) to prevent the tissue from drying out. After the excess fat and ligamentous tissue

were removed, the meniscus was dissected into small pieces to enable more efficient digestion.

Cells were isolated separately from cartilage and meniscus using pronase and collagenase

digestion as previously described (21). The cells were cultured in 10% serum DMEM-F12

media until confluent. Cultures were serum-starved overnight before media collection.

A 4mm biopsy punch was used to harvest human cartilage explants from the cartilage

pieces that had been dissected away from the subchondral bone. Three explants per donor were

pooled in one well and cultured in 10% serum DMEM-F12 media for approximately 72 hours.

The explants were serum-starved for 48 hours before media collection. The explants were

digested in papain, and the DNA content was quantified using the PicoGreen kit (Life

Technologies) as previously described (22).

Experimental animals. The mice used for these studies were housed and maintained

according to the IACUC guidelines at the respective institutions where each study was

performed (The University of Michigan, Wake Forest School of Medicine, and The University of

North Carolina at Chapel Hill). Knee joints from Mif-/- and wild-type (WT) mice for the aging

experiment were from a previously reported study (19). For the surgically-induced OA studies,

the Mif-/- colony was bred on the SV129/C57Bl/6 mixed background. The colony was

maintained by heterozygous breeding, so the Mif-/- and WT mice used in these studies were age-

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and strain-matched littermates. The C57Bl/6 mice used in the neutralizing antibody study were

purchased directly from Jackson Labs.

ELISA. MIF content in serum-free conditioned media was quantified by solid-phase

ELISA (R&D Systems) according to the manufacturer’s protocol. Due to the high level of MIF

in the media, the media samples were diluted 1:25 in the assay.

MIF immunostaining. Human normal and OA cartilage sections were a kind gift from

Martin Lotz (Scripps Research Institute, La Jolla, CA). Human cartilage sections and mid-

coronal sections from 12-month old wild-type and Mif-/- mouse knee joints were immunostained

with anti-MIF (Life Technologies). Sections were de-paraffinized and rehydrated in serial

ethanol washes followed by antigen retrieval in citrate buffer. Sections were first blocked with

3% H2O2 (Fisher Scientific) and then with Protein Block (Dako). The primary antibody was

diluted in antibody diluent (Dako) and incubated on the sections overnight at 4°C. The

following day, the sections were incubated with HRP-linked secondary antibody (Dako) and

developed with the DAB chromagen (Dako). The sections were counterstained in Mayer’s

Hematoxylin (Sigma) and then dehydrated in serial ethanol washes.

DMM procedure. The destabilization of the medial meniscus (DMM) procedure was

performed in 12-week old male Mif-/- and WT mice as described previously (23). This

procedure induces OA by transecting the medial meniscotibial ligament (MMTL). For the sham

surgery, the joint was opened and visualized, but the MMTL was not cut. We randomly assigned

14-16 mice to each surgical group and to each of the anti-MIF treatment and control groups

(described below). The number of mice chosen for the present study was based on a power

calculation using data from a previously published DMM and sham control experiment from our

group that included 12-week old male C57BL/6 mice (23) and found that a sample size of 12

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mice per group would provide greater than 80% power to detect at least a 50% difference in

articular cartilage structure scores between groups. After the surgical procedure, the mice were

monitored closely for signs of apparent pain or adverse effects of the procedure. The mice were

maintained in normal housing conditions and allowed to exercise through normal activities. The

mice were euthanized 10 weeks post-surgery, and the hind-limbs were collected in 10% formalin

(Fisher Scientific) for histological analysis.

In the anti-MIF study, a neutralizing murine anti-MIF IgG1 (clone NIHIIID.9) and

isotype control antibody were purified from ascites fluid using Protein A/G spin columns

(Thermo Scientific). Following isolation, the antibodies were dialyzed overnight in DPBS

(Lonza) and then filter-concentrated by centrifugation (Millipore). The antibody concentration

was measured by the BCA assay (Thermo Scientific). After DMM surgery, mice were allowed

to recover for five days before antibody treatment began. The antibodies were administered by

IP injection for the duration of the study at a dose (20 mg/kg twice per week) previously shown

to neutralize MIF in vivo (24). The mice were euthanized 10 weeks after surgery, and the legs

were collected in formalin for histological analysis. Because repeated handling and injections

can cause pain or other adverse effects which would be reflected with a decrease in food intake,

the body weight of each mouse was monitored throughout the course of the study. All mice

gained weight consistently over the course of the study, and there was no difference in the

average body weights of each group at any time point during the study (data not shown).

Histology processing and OA grading. The formalin-fixed hind-limbs were transferred

to 70% ethanol, and the excess soft tissue was removed. The samples were decalcified in 19%

EDTA (Fisher Scientific), and the intact joints were embedded in paraffin. The joints were

sectioned along the coronal plane at 4µm. Mid-coronal sections were stained with hematoxylin

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and eosin (H&E) and scored using the articular cartilage structure (ACS) score developed by

McNulty et al (25). This system scored the integrity of the articular cartilage on a scale of 0-12,

where 0 represents normal healthy cartilage and 12 represents full-thickness loss of the articular

cartilage across more than two-thirds of the surface scored. Adjacent mid-coronal sections were

also stained with Safranin O and fast green and scored using the Saf-O score developed by

McNulty et al (25). The Saf-O system scored the proteoglycan content of the articular cartilage

on a scale of 0-12, where 0 represents uniform staining of healthy cartilage and 12 represents

complete loss of staining across more than two-thirds of the surface. The Saf-O score is more

sensitive to the mild to moderate lesions that result from the DMM procedure, as it relies on

proteoglycan loss from the matrix vs. fibrillation and loss of cartilage. Therefore, it (rather than

the ACS scoring system) was used to score OA in those studies. Additional joint measurements

were made using the OsteoMeasure histomorphometry system (OsteoMetrics) as described (25).

The additional parameters measured were: articular cartilage area and thickness, subchondral

bone area and thickness, number of viable chondrocytes, area of chondrocyte necrosis, and

calcified cartilage area and thickness.

Osteophyte assessment. Osteophytes were graded using a system that was modified

from a previously published study (26). Osteophytes were graded on a scale of 0-3 as follows: 0

= no osteophyte, 1 = questionable/borderline osteophyte, 2 = small osteophyte, and 3 = large

osteophyte. The summed osteophyte scores for the medial tibial plateau and the medial femoral

condyle are presented.

Synovial assessment. Coronal sections were immunostained with anti-S100A8 (kindly

provided by Johannes Roth) which has been used as a method to detect synovitis with synovial

hyperplasia in mouse joints with OA (27). Immunohistochemistry was performed as above

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except that proteinase K was used for antigen retrieval. Synovial hyperplasia was graded on a

single mid-coronal section from each mouse using a scale of 0-3 as follows: 0 = 1-3 cell layers

in synovium, 1 = 4-6 cell layers, 2 = 7-9 cell layers, and 3 = 10 or more cell layers. The medial

and lateral compartments of the joint were scored separately, and the sum of the two scores is

presented.

MicroCT analysis. The knee joints from 8-week old (n=3 each Mif-/- and WT) and 12-

month old (n=4 Mif-/- and n=5 WT) male mice were scanned with 10 µm resolution by microCT

(µct80; Scanco Medical AG, Brüttisellen, Switzerland) as previously described (28). Bone

morphometric parameters were measured in the trabecular bone of the proximal metaphysis of

the tibia using Scanco software (Scanco Medical AG, Brüttisellen, Switzerland). The region of

interest was consistently defined to begin inferior to the growth plate and extended for 1.00mm

distally. Bone image data were rotated as necessary to adjust for any vertical alignments. The

following histomorphometric trabecular bone parameters (29) were analyzed using the same

threshold for each animal: bone volume fraction, tissue mineral density, connectivity density,

trabecular number, trabecular separation, and trabecular thickness.

Statistical Analysis. All statistical analyses were performed using GraphPad Prism 6

with one exception: OA histomorphometry measurements were analyzed using SPSS, as noted.

Data are presented graphically as individual data points with horizontal lines representing the

mean of each group. MIF ELISA samples were collected from monolayer cultures of cells or

explant cultures from unique human tissue donors (n=7-16 donors/group) and analyzed by

unpaired t-test within each tissue type. OA severity (ACS and Saf-O scores) was analyzed from

individual mice (n=13-16 mice/group) by Kruskal-Wallis nonparametric one-way ANOVA

followed by Dunn’s multiple comparisons test. Histomorphometric measurements were

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analyzed by heteroscedastic t-test. Synovial hyperplasia was evaluated in a subset of mice from

the 12- and 22-month old groups (n=5-11 mice/group) and analyzed by Kruskal-Wallis

nonparametric one-way ANOVA followed by Dunn’s multiple comparisons test. Bone

parameters were measured in individual mice (n=3-5 mice/group) and analyzed by two-way

ANOVA followed by Tukey’s multiple comparisons post-hoc test.

RESULTS

Human OA chondrocytes secrete higher levels of MIF than normal chondrocytes.

Due to previously published results showing that MIF is present in higher levels in OA synovial

fluid than in normal synovial fluid (14, 18), the amount of MIF secreted by joint tissue cells was

measured to determine which tissues could be a source of MIF within the joint. Conditioned

media samples were collected from unstimulated monolayer cultures of chondrocytes and

meniscal cells and from unstimulated cartilage explant cultures, and the MIF protein level in the

media was measured by ELISA (Fig. 1A and 1B). Chondrocytes isolated from OA cartilage

secreted an average of 88.76±13.14ng/mL of MIF over 16 hours, which was significantly higher

(p=0.0005) than that produced by chondrocytes from normal cartilage, which secreted an average

of 28.23±7.78ng/mL of MIF. Cells from OA meniscus secreted an average of

47.62±11.73ng/mL of MIF, which was not significantly different from cells isolated from normal

meniscus (31.65±5.54ng/mL). MIF in conditioned media from OA synovial fibroblasts was not

detectable (data not shown).

OA cartilage explants released an average of 167.9±55.92pg MIF/ng DNA, which was

significantly higher (p=0.0168) than that produced by normal cartilage explants, which released

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an average of 59.21±10.87pg MIF/ng DNA. MIF levels in human cartilage were visualized by

immunostaining. MIF was localized to chondrocytes in sections from both normal (Fig. 1C) and

OA donors (Fig. 1D) without significant differences in immunopositivity suggesting that the

differences noted in cultured cells and explants were due to differences in MIF release into the

media.

Mif deletion in mice reduces the severity of naturally occurring OA with age. In

order to determine if MIF contributes to the development of OA in vivo, the stifle joints from 12-

and 22-month old male Mif-/- and WT mice were analyzed for OA severity. Histological

evaluation of hematoxylin and eosin (H&E) sections revealed characteristic OA changes in the

joints of the WT mice at both ages, including degradation and loss of the articular cartilage,

thickening of the subchondral bone, and osteophyte formation (Fig. 2A). In contrast, the joints

of the Mif-/- mice displayed more healthy appearing articular cartilage, normal thickness of the

subchondral bone, and no osteophyte formation. Anti-MIF immunostaining in a WT section

showed MIF expression in the meniscus and the articular cartilage (Fig. 2A). As expected, there

was a lack of staining for MIF in the sections from Mif-/- mice.

OA severity in the 12- and 22-month old Mif-/- and WT mice, graded using the articular

cartilage structure (ACS) score, revealed significantly higher ACS scores in the WT relative to

the Mif-/- mice at both 12 and 22 months of age (Fig. 2B). WT mice at 12 and 22 months

developed significantly larger osteophytes than the Mif-/- mice (Fig. 2C). The joints of the 12-

and 22-month old Mif-/- and WT mice were further analyzed using histomorphometric analysis

(Table 1). In both age groups, the Mif-/- mice maintained significantly greater articular cartilage

area and thickness and more viable chondrocytes than the WT mice. Subchondral bone changes

were also evident between the genotypes. The subchondral bone area and thickness were

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significantly greater in WT than in Mif-/- mice. The only two parameters which were not

significantly different between Mif-/- and WT were the area of chondrocyte necrosis and the

calcified cartilage area.

Synovitis with synovial hyperplasia is another characteristic of OA. S100A8 is an

alarmin protein that is produced by synovial cells as well as activated macrophages and has been

shown to correlate with synovitis (27, 30). Here, immunohistochemistry was performed using

anti-S100A8 on coronal sections from 12- and 22-month old WT and Mif-/- mice (representative

images are shown in Fig. 3A and 3B). In both genotypes, immunopositivity was evident

throughout the joint – in the articular cartilage, the meniscus, and the synovium. Specifically in

the WT joints, S100A8 immunopositivity was strong in both the cells and matrix of the

synovium. As shown in Fig. 3A, S100A8 immunopositivity correlated with areas of synovial

hyperplasia and thickening of the synovial lining. In contrast, the S100A8 synovial

immunostaining results in the Mif-/- mice revealed minimal synovial hyperplasia. Based on the

described synovial scoring system, there was no difference in the synovial hyperplasia between

the 12-month old WT and Mif-/- mice, while the 22-month old WT mice exhibited more severe

synovial hyperplasia than the Mif-/- mice (Fig. 3C). Additionally, the severity of synovial

hyperplasia increased between 12 and 22 months in the WT mice.

Early bone differences in mice with Mif deletion are not maintained with age.

Because of conflicting reports of bone density differences in Mif-/- mice compared to WT mice

(31, 32) and the possibility that bone density differences could influence the development of OA

(33), we measured trabecular bone parameters by µCT scans of the proximal metaphysis of the

tibia from 8-week old and 12-month old male Mif-/- and WT mice. The 8-week old male Mif-/-

mice had significantly greater bone volume fraction, tissue mineral density, and connectivity

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density than the age-matched WT mice. These differences were not maintained with age, as

there were no significant differences in any bone parameters between 12-month old male Mif-/-

and WT mice. Bone density parameters were also measured in female mice of the same ages,

but there were no differences in these parameters between Mif-/- and WT mice in either age

group (data not shown).

Mif deletion or treatment with a MIF neutralizing antibody does not reduce the

severity of surgically-induced OA in young adult mice. We next wanted to determine if MIF

was required for the development of surgically-induced OA in young adult mice. Here, we used

the destabilization of the medial meniscus (DMM) surgical model to induce OA in 12-week old

male Mif-/- and WT mice and evaluated the severity of cartilage lesions 10 weeks after surgery.

Compared to the sham operated controls, both the WT and Mif-/- mice developed significant

cartilage lesions; however, in contrast to the data on natural age-related OA, there was no

difference in severity between the two groups (Fig. 5A). Both the WT and Mif-/- DMM mice

developed significantly larger osteophytes than the sham mice, but there was no difference

between the two groups (Fig 5B).

We also determined if inhibition of MIF systemically, using a neutralizing antibody,

would alter the severity of surgically-induced OA. Twelve-week old male C57Bl/6 mice

underwent the DMM procedure and were then treated for ten weeks with either a MIF

neutralizing antibody or an IgG control antibody. The dose of anti-MIF used here was sufficient

to inhibit MIF activity in a previous study (24). OA severity was analyzed ten weeks after

surgery (Fig. 5C). Due to limitations in the amount of MIF antibody available and our previous

data indicating that a sham control group did not develop OA changes, the contralateral legs

were scored as the control group in this study. The DMM limbs of the control IgG-treated group

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and the anti-MIF-treated group developed significantly more severe OA than the contralateral

limbs, and there was no difference in OA severity between the two treatment groups. The DMM

limbs of both the IgG and MIF treatment groups developed significantly larger osteophytes than

the contralateral limbs (Fig 5D).

DISCUSSION

Although OA is characterized by common pathologic changes within affected joints, the

pathways that lead to OA can vary depending on the inciting factors. Here, we demonstrate that

deletion of the pro-inflammatory cytokine MIF protects mice from developing naturally

occurring age-related OA but not from developing injury-induced OA. These findings are in

stark contrast to previous studies on mice with deletion of the pro-inflammatory cytokine IL-6

where age-related OA was more severe in the IL-6 knockouts (34) while they were protected

from injury-induced OA using the same DMM model used in our MIF study (35). Other studies

have also shown different effects when age-related and injury-induced OA were evaluated in

mice with specific gene deletions including Mmp3 which, similar to our results, had less age-

related OA but no effects on OA severity in an injury model (reviewed in (36)). These studies

emphasize the need to consider more than one model of OA when determining the role of a

particular factor and indicate that the successful treatment of age-related OA and post-traumatic

OA may require different targets.

The aged Mif knockouts not only exhibited less articular cartilage damage compared to

age-matched wild types but also less synovial hyperplasia and fewer OA bone changes, including

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osteophytes. Synovial inflammation is evident in over 60% of clinical cases of OA as measured

by MRI (37). Synovial inflammation is a key source of pain in OA due to macrophage

infiltration and increased vascularization and innervation of the synovium (38). We used the

alarmin S100A8 as a marker of synovial changes due to its strong correlation with synovial

hyperplasia and synovitis as well as its association with macrophage infiltration in the synovium

(27, 30). However, while there is a clear correlation between the Mif genotype, synovial

hyperplasia, and OA severity, it is difficult to determine if lack of MIF resulted in less synovial

involvement and this contributed to cartilage protection or if less cartilage damage in the Mif-/-

mice resulted in less synovial hyperplasia. The DMM model effectively induces OA changes in

the cartilage and bone, but minimal synovitis has been observed in this model (39). We also did

not observe significant synovitis or synovial hyperplasia in our DMM mice (data not shown)

suggesting that the differential effect of Mif knockout on age-related OA and DMM-induced OA

may be due to a differential role of the synovium. This would be consistent with studies that

have shown that MIF contributes to synovitis in models of inflammatory arthritis (40). As MIF

is constitutively expressed in the joint, it is possible that it exerts an age-related deleterious

action on joint homeostasis but is not important in the acute setting of joint injury when the

expression of other inflammatory mediators prevail.

The finding that the ACS scores in the wild-type mice did not change significantly

between 12- and 22-months was unexpected. Since this was a cross-sectional study it is not

possible to determine if this was due to a lack of progression of the cartilage changes or just a

chance occurrence in two different sets of mice. However, we did note that there was a

significant decrease in the subchondral bone area between the 12- and 22-month old WT mice,

and there was a trend towards a significant decrease in the articular cartilage area between the

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two groups, but we did not note differences in the number of viable chondrocytes or in the area

of chondrocyte necrosis. These aging changes in the mice are similar to aging changes in

humans where there is a thinning of the articular cartilage (2), a decrease in the number of viable

chondrocytes (41), and an overall increase in bone turnover (1).

There is some controversy over the role of MIF in bone. Studies have shown that MIF

inhibits osteoclastogenesis through activation of the tyrosine kinase Lyn (42), and that Mif-/-

mice have significantly less bone volume than WT mice (32). Alternatively, MIF has been

shown to be necessary for osteoclastogenesis in a mouse model of rheumatoid arthritis and Mif-/-

mice were protected from development of bone erosions in this model (15). Because increased

bone density has been shown to be a potential risk factor for incident OA while increased bone

turnover may promote progression (43, 44), we examined bone density in young and older adult

Mif-/- mice. We found that the BV/TV and other bone parameters were higher in young Mif-/-

mice compared to WT mice, but these early bone differences were not maintained with age.

Therefore, it is unlikely that these early bone density differences were responsible for the

reduced OA severity seen in the older adult Mif-/- mice.

It is not clear from the present study if Mif deletion had a direct effect on the articular

chondrocytes that could explain less severe age-related OA. Immunohistochemistry results

revealed that chondrocytes, as well as meniscal cells, were immunopositive for MIF and released

MIF into the media when cultured. Similar intracellular staining and release has been seen with

other cell types such as macrophages (12) and RA synovial fibroblasts (14). Chondrocytes, but

not meniscal cells, from OA joints secreted more MIF than cells from normal joints. However,

we could not detect an effect of extracellular MIF on joint tissue cells. We performed a series of

in vitro studies using recombinant MIF in doses up to 1 µg/mL and could not detect an increase

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in ERK, p38, or JNK MAP kinase activation or increase in MMP production in cultures of

human chondrocytes, meniscal cells, or synovial fibroblasts (supplemental Fig.1).

These in vitro results with joint tissue cells are contrary to published studies with other

cell types that reported recombinant MIF-stimulated phosphorylation of the ERK MAP kinase in

Raji cells (a B cell line) (9) and in primary rat osteoblasts (45). Additional published studies

show recombinant MIF-stimulated production of MMP-1 and MMP-3 production in synovial

fibroblasts isolated from OA and RA patients (13) as well as MMP-9 and MMP-13 production in

primary rat osteoblasts (45). However, those studies required doses of MIF up to 10 µg/mL to

detect a response that far exceeds the amount of MIF measured in synovial fluid, which ranges

from 3 to 19 ng/mL (14, 18), or the amount of MIF we measured in conditioned media from

confluent chondrocyte and meniscal cell monolayers, which ranged from 1 to 140 ng/ml. We

examined chondrocytes for the presence of CD74, which is the primary receptor for MIF, but

could not detect significant levels on the cell surface by flow cytometry (unpublished results).

These findings suggest that chondrocytes produce and secrete MIF but that it does not have an

autocrine effect on cartilage. MIF may instead promote OA by promoting macrophage

infiltration into the joint, which would explain the reduced synovial hyperplasia observed in the

aged mice with MIF deleted. MIF also has been described to exert intracellular functions by

intracytoplasmic interaction with the COP9 signalosome that influences cell cycle progression

(46). Accordingly, the distinction we observed between the effects Mif deletion versus

immunoneutralization may be accounted for by a strictly intracellular role for MIF in

chondrocytes.

Taken together, the results presented here indicate that the lack of MIF has a strong

protective effect on naturally occurring age-related OA. As the percentage of adults over the age

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of 65 continues to increase, the prevalence of aging-related diseases, including OA, is also

increasing (47). MIF is encoded in a functionally polymorphic locus that occurs commonly in

the population (48), and our data prompt investigation into the potential role of variant MIF

alleles in OA incidence or progression. Therapies that are able to stop or slow the progression of

diseases of aging are necessary in order to improve the quality of life and to extend the healthy

lifespan of the aging population.

ACKNOWLEDGEMENTS

We thank the Gift of Hope Tissue and Organ Donor Network, Dr. Susan Chubinskaya,

and the donor families for providing normal donor tissue. We also thank the Department of

Orthopaedic Surgery at Wake Forest Baptist Health and the University of North Carolina for

providing osteoarthritic tissue. We thank Dr. Tom Smith (Department of Orthopaedic Surgery,

Wake Forest School of Medicine) and Kathryn Kelley (Thurston Arthritis Research Center,

University of North Carolina at Chapel Hill) for their assistance in performing the DMM

surgeries as well as Melissa Roy, Dr. Alexandra Armstrong, and Dr. Laura Pritzker (University

of Minnesota) for their assistance with histological grading/morphometry and Dr. Ted Bateman

and Eric Livingston (University of North Carolina at Chapel Hill) for their assistance with the

microCT. We also thank Dr. Martin Lotz (Scripps Research Institute, La Jolla, CA) for his kind

gift of the human cartilage sections, Dr. Peter van der Kraan (Radboud University Medical

Center, Nijmegen) for his advice regarding our synovial studies, and Dr. Johannes Roth (Institute

of Immunology, University of Münster, Münster) for his kind gift of the S100A8 antibody.

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TABLES

Table 1. Histomorphometric analysis of aged mice with Mif deletion and wild-type controls.

12-month old 22-month old

WT

12- vs

22-

month

olds

Parameter Mif-/-

Mean (SD)

WT

Mean (SD) p-value

Mif-/-

Mean (SD)

WT

Mean (SD) p-value p-value

Art Cart Area

(mm2)

0.064

(0.011)

0.040

(0.016) <0.001

0.064

(0.008)

0.029

(0.018) <0.001 .089

Art Cart

Thickness

(mm)

0.072

(0.013)

0.043

(0.017) <0.001

0.078

(0.009)

0.037

(0.019) <0.001 .339

Subchondral

Bone Area

(mm2)

0.080

(0.016)

0.156

(0.069) 0.002

0.070

(0.025)

0.103

(0.038) 0.009 .024

Subchondral

Bone

Thickness

(mm)

0.059

(0.011)

0.104

(0.046) 0.004

0.070

(0.023)

0.100

(0.037) 0.013 .772

Number of

Viable

Chondrocytes

138.39

(16.15)

74.31

(41.38) <0.001

115.50

(21.98)

64.14

(45.24) 0.001 .549

Area of

chondrocyte

necrosis

(mm2)

0.008

(0.006)

0.006

(0.004) 0.396

0.008

(0.011)

0.007

(0.008) 0.859 0.602

Calcified

cartilage area

(mm2)

0.063

(0.012)

0.059

(0.019) 0.504

0.049

(0.005)

0.053

(0.019) 0.368 .421

Histomorphometry measurements of cartilage (Cart) and bone parameters in the medial tibial

compartment of mid-coronal stifle sections were analyzed using the Osteomeasure

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Histomorphometry Program (OsteoMetrics®). 12-month old WT: n=13; 12-month old Mif-/-:

n=13; 22-month old wild-type (WT): n=14; 22-month old Mif-/-: n=16. Significance differences

determined by heteroscedastic t-test using SPSS.

FIGURE LEGENDS

Figure 1. MIF levels in conditioned media from normal and OA chondrocyte and meniscal cell

cultures and in normal and OA cartilage sections. A. Serum-free medium was collected after 16

hours of culture from unstimulated confluent monolayers of normal and OA chondrocytes and

meniscal cells. MIF protein in media was measured by human MIF ELISA. Horizontal lines on

the graph represent average MIF level for each tissue type; individual data points are shown.

Normal chondrocytes: n=12 donors; OA chondrocytes: n=9 donors; normal meniscus: n=8

donors; OA meniscus: n=9 donors. Statistical significance was evaluated by unpaired t-test

within tissue types. B. Serum-free conditioned medium was collected after 48 hours of culture

from unstimulated human and OA cartilage explants. MIF protein in media was measured by

human MIF ELISA and was normalized to the DNA content of the explants. Individual data

points are shown. Normal cartilage explants: n=16 donors; OA cartilage explants: n=7 donors.

Statistical significance was evaluated by unpaired t-test, and no outliers were identified by the

Grubbs outlier test. Immunohistochemistry results for MIF in sections of (C) human normal

knee cartilage and (D) human OA knee cartilage.

Figure 2. Mice with Mif deletion are protected from age-related OA. A. Representative

histologic images of knee joint sections in 12-month old Mif-/- and WT mice. (i) WT mouse,

medial tibial plateau (MTP) articular cartilage structure (ACS) score 11. Degradation and loss of

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articular cartilage (arrow), osteophyte formation (arrowhead), and thickening of subchondral

bone (bracket). (H&E) (ii) Mif-/- mouse, ACS score 3. Normal subchondral bone thickness

(bracket). (H&E) (iii) WT mouse section immunostained for MIF. Intracellular MIF is present in

meniscus (arrow) and articular cartilage (arrowhead). (iv) Mif-/- mouse. Lack of MIF

immunopositivity in meniscus (arrow) and cartilage (arrowhead). B. Articular cartilage structure

scores in Mif-/- and WT mice. The medial tibial plateau of mid-coronal stifle sections was

scored using the articular cartilage structure (ACS) score. 12-month old WT: n=13; 12-month

old Mif-/-: n=13; 22-month old WT: n=14; 22-month old Mif-/-: n=16. C. Osteophyte scores in

Mif-/- and WT mice from (B). The summed scores from the medial tibial plateau (MTP) and

medial femoral condyle (MFC) are presented. Horizontal lines on graph represent the mean of

each group. Significant differences were determined by Kruskal-Wallis nonparametric one-way

ANOVA followed by Dunn’s multiple comparisons test.

Figure 3. Synovial hyperplasia is less severe in 22-month old mice with Mif deletion.

Immunohistochemistry results for S100A8 in (A) 22-month old WT mouse, ACS score 11, and

(B) 22-month old Mif-/- mouse, ACS score 0. Arrows indicate regions of strong synovial

S100A8 immunopositivity and synovial hyperplasia. Representative histologic images are

shown. M=meniscus. C. Synovial hyperplasia scores of the medial and lateral compartments

were summed from 12-month old WT (n=5) and Mif-/- (n=5) and from 22-month old WT (n=11)

and Mif-/- (n=9) mice. Horizontal lines on graph represent the mean of each group. Significant

differences were determined by Kruskal-Wallis nonparametric one-way ANOVA followed by

Dunn’s multiple comparisons test.

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Figure 4. µCT analysis of 8-week old and 12-month old male mice with Mif deletion and wild-

type controls. Bone density parameters of the proximal metaphysis of the tibia were analyzed

using Scanco software in 8-week old WT (n=3) and Mif-/- (n=3) and in 12-month old WT (n=4)

and Mif-/- (n=5) mice. A. Bone volume fraction. (BV/TV=bone volume/total volume) B. Tissue

mineral density. (HA=hydroxyapatite) C. Connectivity density. D. Trabecular number. E.

Trabecular separation. F. Trabecular thickness. Horizontal lines on graphs represent the mean

of each group. Significant differences were determined by two-way ANOVA followed by

Tukey’s multiple comparisons post-hoc test.

Figure 5. Inhibition of MIF activity either by gene deletion or by neutralizing antibody does not

protect young adult mice from injury-induced OA. A. Twelve-week old Mif-/- or WT mice

received either the DMM or sham procedure. OA severity was analyzed 10 weeks after surgery

by the Saf-o score. Mif-/- DMM: n=14; Mif-/- sham: n=16; WT DMM: n=16; WT sham: n=13.

B. Osteophytes were measured in the mice from (A) and summed scores from the MTP and

MFC are presented. C. Twelve-week old WT mice received the DMM procedure and were

treated for 10 weeks with either the MIF neutralizing antibody (Anti-MIF) or IgG control

antibody. The contralateral limb was used as the un-operated control. OA severity was analyzed

10 weeks after surgery by the Saf-O score. Anti-MIF DMM: n=15; Anti-MIF contralateral

control: n=15; IgG DMM: n=14; IgG contralateral control: n=14. D. Osteophytes were

measured in the mice from (C) and summed scores from the MTP and MFC are presented.

Horizontal lines on the graph represent the mean of each group. Significant differences were

determined by Kruskal-Wallis nonparametric one-way ANOVA followed by Dunn’s multiple

comparisons test.

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